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J Struct Funct Genomics. 2014 Dec;15(4):191-9. doi: 10.1007/s10969-014-9190-1. Epub 2014 Nov 15.

Development of a hexahistidine-3× FLAG-tandem affinity purification method for endogenous protein complexes in Pichia pastoris.

Author information

1
Department of Supramolecular Biology, Graduate School of Nanobioscience, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi, Yokohama, Kanagawa, 230-0045, Japan.

Abstract

We developed a method for efficient chromosome tagging in Pichia pastoris, using a useful tandem affinity purification (TAP) tag. The TAP tag, designated and used here as the THF tag, contains a thrombin protease cleavage site for removal of the TAP tag and a hexahistidine sequence (6× His) followed by three copies of the FLAG sequence (3× FLAG) for affinity purification. Using this method, THF-tagged RNA polymerases I, II, and III were successfully purified from P. pastoris. The method also enabled us to purify the tagged RNA polymerase II on a large scale, for its crystallization and preliminary X-ray crystallographic analysis. The method described here will be widely useful for the rapid and large-scale preparation of crystallization grade eukaryotic multi-subunit protein complexes.

PMID:
25398586
PMCID:
PMC4237914
DOI:
10.1007/s10969-014-9190-1
[Indexed for MEDLINE]
Free PMC Article

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